Original Article
125
Extraction and Demonstration of Uterotonic Activity
from the Root of Steganotaenia Araliacea Hochst1* 1 2 3 1 1 2FM Goma, , C Ezeala , JNyirenda , D Chuba , L Prashar , N Simfukwe , C Lengwe
1
2Department of Chemistry, School of Natural Sciences; 3Department of Biological Sciences, University of Zambia, Lusaka, Zambia
Department of Physiological Sciences, School of Medicine;
*Corresponding AuthorDr. Fastone M Goma PhD, MBChBDepartment of Physiological Sciences,School of Medicine, University of Zambia, Ridgeway Campus, Lusaka 10101, Zambia; [email protected]@yahoo.co.uk
ABSTRACT
Background: The root of Steganotaenia araliacea
is used for assisting labour in folk medicine. Recent
reports indicate that the root could possess
uterotonic substances.
Objectives: The study aimed to evaluate three
methods for the extraction of uterotonic principles
from the root of S. araliacea growing in Zambia.
Methods: Roots of the plant were collected from
Chongwe District of Zambia. The air-dried roots
were size-reduced, and the powdered material
extracted with hot ethanol, hot distilled water, and
cold distilled water. The solvent extracts were 0concentrated and dried at 110 C. Solutions of the hot
aqueous and cold aqueous extracts were prepared in
distilled water and used for organ bath experiments
to demonstrate uterotonic activities using strips of
pregnant rat uterus. The frequencies and amplitude
of contractile forces were recorded. The amplitudes
were plotted against log concentration of extract
with GraphPad Prism software, and the EC50 values
determined.
Results: The percentage yields were 31.3 % for the
hot aqueous extract, 8.15 % for the ethanolic extract,
and 3.27 % for the cold aqueous extract. The cold
aqueous extract showed higher potency (EC50 of
0.54 mg/ml) compared to the hot aqueous extract
(EC50 of 2.09 mg/ml).
Conclusion: Root extracts of S. araliacea possess
demonstrable uterotonic effects. Extraction of the
roots for this purpose could benefit from preliminary
defatting with organic solvents, followed by
successive extraction with hot and cold water.
INTRODUCTION
The demand for revitalization of health care is ever
on increase, parallel to this is the demand of services
of Traditional Medicines. The use of Traditional
Medicines all over the world is demonstrating to be
phenomenal. Traditional medicine has a great
potential of therapeutic benefits in its contribution to
modern medicine. Presently more than 30% of
marketed modern medicines are derived directly or 1indirectly from medicinal plants .
WHO estimates that 80% of the population in
developing countries depends on traditional 2medicines for their primary health care . The world
market for Traditional Medicines has reached
US$60 million, with annual growth rates between 5
and 15%. Traditional medicinal plants and products
can be targeted for patent claims since they have
become of great interest to the globally organized 3herbal drug and cosmetic industries . Zambia is a
member of World Trade Organization since 1995.
Keywords: Childbirth, extraction methods,
medicinal plant, potency, Steganotaenia araliacea,
uterotonic effect
Medical Journal of Zambia, Vol. 44, No. 2: 125-132 (2017)
Under Article 27 of Trade Related Intellectual
Property Rights Agreement; members will provide
for the protection of plant varieties either by patents
or by an effective sui generis system or by any 4combination thereof . With the Doha declaration on
TRIPS agreement and Public Health, it becomes
important for us to protect our Intellectual Property
Rights in the form of Indigenous Knowledge 5
Systems (IKS) .
IKS generally refers to age-old long-standing
traditions and practices involving wisdom,
knowledge and teachings of communities and 6encompassing traditional technologies .
For the most part, the knowledge is passed down
through generations, usually from designated
persons to others using various modes of
communication like story-telling, rituals, songs or
laws. This framework enables growth and
sustainability of communities which invariably
includes sustaining the health of community
members.
It can, therefore, be said that a portion of what
indigenous knowledge imparts is traditional 7medicine knowledge . These therapeutic practices
have been in existence for hundreds of years before
the development of conventional scientific
medicines e.g. morphine from Papever
somniferum, quinine from Cinchona officinalis,
strychnine from Srychnox nuxvomica and ephedrine 8
from Ephedra sinica .
To preserve these knowledge systems, the regional
office in Brazzaville, responsible for African region
has facilitated activities leading to the present trend
of recognition and development of herbal medicines
in Africa by various international organisations.
This calls for positive response from all the medical
and pharmaceutical research scientists of African
origin. The decade 2001-10 was tagged as 'Decade st
of Traditional Medicine in Africa and 31 August is
celebrated every year as the, 'Traditional Medicine 8
Day' .
Traditional health practitioners (THPs) have used
herbal plants and other remedies for beneficial
effects during pregnancy, to induce labour, in the
removal of retained placenta and management of 9
post-partum bleeding . The effects of these
medicines have been reported informally, mostly in
circumstances where their effects have been rather
excessive causing injury to the uterus (rupture) or
death by peri partum heamorrhage. These
exaggerated responses are suspected to be related to
overdosing since there are no regulated traditional
measurements. This study compared uterotonic
activities in extracts of Steganotaenia araliacea
Hochst obtained using three different solvents and
conditions. The purpose was to optimise the
extraction process for uterotonic principles from the
root.
METHODS
A. Plant collection and identification
The investigators consulted with local people in
Chongwe District to help locate the plant. Root
samples of the plant were collected from the
Chongwe District of Zambia using standard
procedures. The samples were immediately placed
in a black plastic bag to prevent any possible solar
degradation of active compounds. For taxonomic
verification, specimens of the plant were dried by
placing them in a plant press and sun drying for
about three days while changing blotting papers
each day. The plant was identified to species level in
the field using prior knowledge and later confirmed
by physical comparison with specimens in the
herbarium at University of Zambia (UNZA) and by
consulting the taxonomic literature. Voucher
specimens were deposited in the UNZA herbarium.
B. Processing and extraction of the roots
Preliminary Treatment
The roots were freed of soil and debris, and size
reduced using a clean laboratory axe and then air-
dried in the shade for 14 days under blotting paper.
Moisture content was monitored to a constant level
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Medical Journal of Zambia, Vol. 44, No. 2: 125-132 (2017)
of 6 % (wt/wt), as a function of weight loss. A
laboratory blender (Warring Commercial,
Christison Particle Technologies, UK) was used to
reduce the samples further, and the powdered
material stored in Ziploc plastic bag.
Extraction
The powdered sample was subjected to three types
of extraction; soxhlet, hot and cold water extraction.
Soxhlet extraction using ethanol (distilled)
Approximately, 302 g of the powdered sample was
extracted with about 2600 ml of distilled ethanol for
about 10 hrs using soxhlet apparatus. This was
followed by solvent evaporation using a rotary
evaporator (Rotavapor R11, BUCHI, Switzerland) o
at 45 C. The resultant glue-like residue was further o
dried in an oven at 80 C for 48 hours. The resulting o
dry extract was stored in the refrigerator at 4 C.
Hot water extraction
Fifty grams (50.0 g) of the powdered sample was
extracted with 800 ml of boiling distilled water for
10 min. The extract was cooled to room temperature,
and then centrifuged at 4000 rpm for 10 min. The
supernatant was decanted and evaporated to near
dryness using a hot plate, and further dried in an o
oven at 110 C. The dry extract was stored in the o
refrigerator at 4 C until required for analysis.
Cold water extraction
Approximately, 302.16 g of the powdered sample
was soaked in 1,750 ml cold distilled water
overnight. The mixture was centrifuged (4000 rpm
for 10 min) and decanted to obtain a clear solution.
The decanted supernatant was evaporated to near
dryness with a hot plate, and then dried in an oven at o 110 C. The extract was then stored in the
orefrigerator at 4 C.
C. Habituation of the animals and isolation of
uterine muscle
Pregnant Female Wistar albino rats aged 7 weeks and weighing between 180 to 200 g were obtained
from the School of Veterinary Sciences of the University of Zambia, Lusaka. They were housed in standard metal cages, maintained at room
otemperature (20 -24 C), 12 hours light/dark cycles, and allowed free access to water and rodent pellets (obtained from Tiger Animal Feeds Ltd, Lusaka).
D. Demonstration of uterotonic activity on isolated uterine muscle
thOn the 10 day of gestation, the rats were sacrificed by cervical dislocation, immediately dissected and the uteri extracted. Any adherent fat, connective tissues, and the foetuses were removed. The uteri were placed in De Jalon's physiological solution at room temperature, and longitudinal strips (about 2 cm long) were cut out from the cleaned uteri and mounted in the organ baths (AD Instruments) containing about 25 ml De Jalon's solution and
omaintained at about 32 C. The force transducers were connected to PowerLab and LabTutor (AD Instruments). Baseline spontaneous contractions were recorded for 5 minutes. Thereafter, solutions of the cold aqueous extract were added to give final bath concentrations varying from 0.016 mg/ml to 2.048 mg/ml with 3 washes between additions of each concentration. The contractions produced by each concentration were recorded on the Lab Tutor for 5 minutes. Using the same tissue, the above procedure was repeated for the hot aqueous extract. The procedure was not carried out for the alcoholic extract as it failed to dissolve in water or saline. Oxytocin was used as positive control; it also served to demonstrate sustained viability of the isolated uterine tissues during the experiments.
E. Data Analysis
The contractions of the uterine muscle produced by graded doses of the hot and cold crude aqueous extracts were analysed to determine the frequency (number of contractions per minute) and magnitude of the force of contraction (the difference between the peak and baseline). Prism GraphPad software (version 7) was used to plot the response against the logarithm of concentrations of the extract. The EC 50
values and Hill Slope factors were determined.
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Medical Journal of Zambia, Vol. 44, No. 2: 125-132 (2017)
RESULTS
Description of the extracts
The alcoholic extract yielded a reddish-brown oily
viscous residue. This could not dissolve in water or
normal saline and was not analysed further. The
cold-water extract gave a brownish-black gummy
residue that was readily soluble in water. This was
reconstituted with cold distilled water and used for
the isolated organ experiments. The hot extract was
a semi-solid dark-brown residue that was also
soluble in water. After reconstitution with distilled
water, it was also used for the organ bath studies.
Figure 1: Physical appearance of: a. Hot-
ethanolic extract, b. Hot-water extract, and c.
Cold-water extract
The Percentage Yield
Hot water extract of root exhibited highest yield
(31.3%) followed by ethanolic Soxhlet extract
(8.15%). The cold water extract showed the lowest
yield of 3.27%.
Table 1: Percentage yield of crude extracts of root
of Steganotaenia araliaceais Hochst
Organ Bath Studies
The cold aqueous extract markedly increased the
amplitude and frequencies of contractions of the
uterine muscles in a concentration dependent
manner, with the lowest tested dose, 0.016 mg/ml,
producing demonstrable increase in contraction
frequency. The hot aqueous extract produced
observable increases in the frequencies and
amplitudes of contractions of the uterine smooth
muscle, but the effects were smaller than the effects
of similar concentrations of the cold extract.
Oxytocin produced a much higher effect.
Figure 2: Baseline spontaneous rhythmic
contractions of the rat uterine smooth muscle
a. b.
c.
Soxhlet Hot water Cold water
Mass of powder (g) 302.0 50.0 302.16
Mass of extract (g) 24.62 15.65 9.87
Yield (%) 8.15 31.30 3.27
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Medical Journal of Zambia, Vol. 44, No. 2: 125-132 (2017)
Figure 3: Contractions induced by different concentrations of the cold aqueous extract
(Final bath concentrations of the cold aqueous extract were a) 0.016 mg/ml, b) 0.064
mg/ml, c) 0.250 mg/ml, d) 0.512 mg/ml, e) 1.024 mg/ml and f) 2.048 mg/ml, respectively).
Figure 4: Contractions induced by different concentrations of the hot aqueous extract
( T h e f i n a l b a t h concentrations of the hot aqueous extract were a) 0.064 mg/ml, b) 0.128 mg/ml, c) 1.024 mg/ml and d) 2.048 mg/ml, respectively).
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Medical Journal of Zambia, Vol. 44, No. 2: 125-132 (2017)
Figure 5: Contractions induced by 5 iu/ml of
oxytocin
Figure 6: Concentration (Dose) -response curves
for the hot and cold extracts.
0.0 0.5 1.0 1.5 2.0 2.50.00
0.05
0.10
0.15
0.20
0.25
Log (DoseX100)
Fo
rc
e
Cold Ext Hot Ext
Concentration (Dose) - Effect Analysis
Quantitative analysis of the concentration –
response relationships showed that the cold extract
was more potent than the hot extract with a lower
EC of 0.58mg/ml compared to EC of 1.06 mg/ml 50 50
for the hot extract. The semi-log plot of the cold
extract was completely sigmoidal . Plot of the hot
extract was remarkably sigmoid; however, outlying
points were removed in order to construct a more
representative curve from which the EC was 50
calculated.
(Log (agonist) vs. response -- Variable slope (four
parameters), EC50 and Hill Slope factor for cold
extract were 0.5428 and 2.09, respectively)
Figure 7: Modified concentration-response curve
for the hot extract
(Log (agonist) vs. response -- Variable slope (four
parameters) reanalysed omitting outlying points,
EC and Hill Slope factor, for hot extract, were 1.06 50
and 0.76, respectively)
DISCUSSION
Extraction of S. araliacea root with ethanol, hot and
cold water resulted in the hot extract having the
highest percentage yield of the extract. The ethanolic
extract, with a moderate percentage yield (8.15 %)
was oily, suggesting that the root contains a
reasonable quantity of fatty material. Defatting of the
powdered root with organic solvents as a preliminary
step in the processing of the root for analysis of its 10 11
bioactivity is recommended , . Furthermore, the oil
from the root of this plant could possess properties
that may be useful medicinally and/or industrially.
Plant oils are widely used in medicine, cosmetology,
aromatherapy, biofuel production, and other 12, 13, 14, 15, 16
industrial applications . The study therefore
recommends further extraction and distillation of the
root oils and investigation of their potential
usefulness in these areas.
0.0 0.5 1.0 1.5 2.0 2.50.00
0.02
0.04
0.06
0.08
Log (DoseX100)
Fo
rc
e
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Medical Journal of Zambia, Vol. 44, No. 2: 125-132 (2017)
The hot aqueous extract showed the highest
percentage yield, but, uterotonic bioactivity was
higher with the cold aqueous extract, which
demonstrated a much higher potency in the organ
bath studies. This finding probably suggests that hot
water extraction removed significant quantities of
substances that have no uterotonic activities 17
(artefacts), leaving a purer cold aqueous extract .
Based on these observations on the percentage
yields and the bioactivities of the extracts, the study
further emphasizes that extraction processes for the
recovery of uterotonic principles from the root of S.
araliacea could benefit from preliminary defatting
of the powdered material with organic solvents. Hot
water extraction to remove artefacts could follow
this step, before cold aqueous extraction of the
biologically active materials from the root.
The study confirms the presence of uterotonic
principles in the root extract. This observation is 18consistent with previous report . Importantly, it has
provided scientific justification for the use of the
root by traditional practitioners in maternal
healthcare and specifically for assisting labour.
Furthermore, the extract could provide a lead for the
development of an orally active drug that could
serve in obstetrics for assisting women with
prolonged labour. Its further development should
include purification, identification, and chemical
characterization of the active principles. These steps
should precede preclinical toxicology, safety
pharmacology, and clinical tests in human and/or
large animal subjects. The material can also be
useful for medical abortion for pregnant women at 19, 20risk .
Induction of uterine smooth muscle contraction
involves complex interplay of different signalling
mechanisms that include neuronal, endocrine,
mechanical, and metabolic processes. Current
evidence shows that pathways mediated by
acetylcholine, oxytocin, histamine, and
prostaglandins may be involved, while calcium and
other divalent cations and inositol triphosphate play 21, 22
roles as second messengers . The exact molecular
mechanism by which the constituents of S.
araliacea interact with these physiological
processes to produce uterine contraction is not
obvious. Chemical characterization of the extract
and in depth pharmacological studies to elucidate
these physiological interactions is in process.
In conclusion, extraction of powdered root of S.
araliacea with ethanol, hot water and cold water
yielded crude extracts with significant uterotonic
activities. The cold aqueous extract demonstrated
higher uterotonic potency than the hot water extract.
This article recommends preliminary defatting of
the powder with organic solvents to remove the oils,
followed by hot-water extraction to remove
artefacts that are not uterotonic, before final cold-
water extraction of the active ingredients. The
product obtained by this process showed great
potential for development into a clinically useful
drug for obstetric use.
This is work in progress, and much more work is
ongoing to isolate and chemically characterise the
active principles and possibly translate this product
to the bench side.
ACKNOWLEDGEMENT
This work was supported by a research grant from
National Science & Technology Council (NSTC),
through the Ministry of Science &Technology of
Zambia.
DECLARATION OF INTEREST
The authors declare no conflict of interest associated
with this work and have no relevant affiliations or
financial involvement with any organization or
entity with a financial interest in or financial conflict
with the subject matter or materials discussed in this
write-up. This includes employers, consultancies,
honoraria, patents (received or pending), expert
testimony, stock ownership, or royalties.
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